Microsecond MD Simulations of the Plexin-B1 RBD: 2. N-H Probability Densities and Conformational Entropy in Ligand-Free, Rac1-Bound, and Dimer RBD
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Updated: Sep 1, 2025
15N CPMG Relaxation Dispersion for the Investigation of Protein Conformational Dynamics on the µs-ms Timescale
Published on: April 19, 2021
Netanel Mendelman1, Yaron Pshetitsky1, Zhenlu Li2
1The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel.
Abstract:
Orientational probability densities, Peq = exp(-u) (u, local potential), of bond-vectors in proteins provide information on structural flexibility. The related conformational entropy, S = -∫Peq(ln Peq)dΩ - ln ∫dΩ, provides the entropic contribution to the free energy of the physical/biological process studied. We have developed a new method for deriving Peq and S from MD simulations, using the N-H bond as probe. Recently we used it to study the dimerization of the Rho GTPase binding domain of Plexin-B1 (RBD). Here we use it to study RBD binding to the small GTPase Rac1. In both cases 1 μs MD simulations have been employed. The RBD has the ubiquitin fold with four mostly long loops. L3 is associated with GTPase binding, L4 with RBD dimerization, L2 participates in interdomain interactions, and L1 has not been associated with function. We find that RBD-Rac1 binding renders L1, L3, and L4 more rigid and the turns β2/α1 and α2/β5 more flexible. By comparison, RBD dimerization renders L4 more rigid, and the α-helices, the β-strands, and L2 more flexible. The rigidity of L1 in RBDRAC is consistent with L1-L3 contacts seen in previous MD simulations. The analysis of the L3-loop reveals two states of distinct flexibility which we associate with involvement in slow conformational exchange processes differing in their rates. Overall, the N-H bonds make an unfavorable entropic contribution of (5.9 ± 0.9) kJ/mol to the free energy of RBD-Rac1 binding; they were found to make a favorably contribution of (-7.0 ± 0.7) kJ/mol to the free energy of RBD dimerization. In summary, the present study provides a new perspective on the impact of Rac1 binding and dimerization on the flexibility characteristics of the RBD. Further studies are stimulated by the results of this work.
Insights
This study reveals how Rac1 binding impacts protein structural flexibility, showing increased rigidity in key loops and turns. This binding contributes unfavorably to the free energy, unlike dimerization which has a favorable entropic contribution.
Area of Science:
- Structural Biology
- Computational Biophysics
- Protein Dynamics
Background:
- Protein flexibility is crucial for biological function and can be quantified using orientational probability densities (P_eq) of bond vectors.
- Conformational entropy (S) derived from P_eq provides insights into the entropic contribution to free energy changes in biological processes.
- The Rho GTPase binding domain (RBD) of Plexin-B1 plays a role in cellular signaling and undergoes conformational changes upon binding to GTPases.
Purpose of the Study:
- To develop and apply a novel method for deriving protein orientational probability densities (P_eq) and conformational entropy (S) from molecular dynamics (MD) simulations.
- To investigate the impact of small GTPase Rac1 binding on the structural flexibility of the Plexin-B1 RBD.
- To compare the effects of Rac1 binding with previously studied RBD dimerization on protein flexibility and entropic contributions.
Main Methods:
- Utilized a new method employing N-H bonds as probes to derive P_eq and S from 1 μs MD simulations.
- Analyzed changes in loop and turn flexibility within the RBD upon binding to Rac1.
- Quantified the entropic contribution of N-H bonds to the free energy of RBD-Rac1 binding and RBD dimerization.
Main Results:
- RBD-Rac1 binding increased rigidity in loops L1, L3, and L4, while enhancing flexibility in turns β2/α1 and α2/β5.
- RBD dimerization, in contrast, increased rigidity in L4 and flexibility in α-helices, β-strands, and L2.
- RBD-Rac1 binding resulted in an unfavorable entropic contribution of (5.9 ± 0.9) kJ/mol, whereas RBD dimerization yielded a favorable contribution of (-7.0 ± 0.7) kJ/mol.
Conclusions:
- Rac1 binding significantly alters the flexibility profile of the Plexin-B1 RBD, impacting specific loops and turns differently than dimerization.
- The observed rigidity of L1 upon Rac1 binding correlates with previously identified L1-L3 contacts.
- The study provides a new perspective on GTPase binding effects on protein dynamics and highlights distinct entropic contributions to binding and dimerization.
